Bbtechnol. RW. 1991, 7, 237-245

Long-Term in Vitro Function of Adult Hepatocytes in a Collagen Sandwich Configuration James C. Y. Dum> Ronald G. Tompkins,*,+and Martin L. Yarmush’BtJ Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, and Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, New Jersey 08854

In an effort to reconstruct the cellular polarity normally found in the liver, adult rat hepatocytes were sandwiched between two layers of hydrated rat tail tendon collagen matrix. Functionally, sandwiched hepatocytes maintained the secretion of albumin, transferrin, fibrinogen, bile acids, and urea for at least 6 weeks, whereas cells cultured on a single layer of collagen gel ceased such secretion in 1-2 weeks. After 1 week of culture on a single layer of collagen gel, hepatocytes could still recover these lost functions when a second layer of collagen gel was applied. The exact nature of the substrate for constructing the sandwich system appeared to be unimportant as long as it allowed cellular attachment. Hepatocytes cultured in the sandwich system appeared to maintain a distribution of actin filaments similar to the in vivo state, whereas cells cultured on a single layer of collagen gel showed abnormal formation of stress fibers. These studies suggest that simple manipulations of the configuration of extracellular elements can dramatically alter the behavior of cultured hepatocytes.

Introduction Hepatocytes perform many complex functions critical to the well-being of the organism, including metabolism, secretion, excretion, detoxification, and storage. There has been much interest in using hepatocytes for the studies of gene expression,regeneration,and artificialliver support (8,14,26,27,46,47). The feasibility of these endeavors depends on the ability to maintain and to manipulate hepatocytes in vitro. Previous studies that attempted to maintain long-term cultures of functional hepatocytes had employed hormonally defined media, dimethyl sulfoxide, coculture, or complex matrices (1,2,9,15,16,20,21,32,33,39,40,42, 43). The culture configuration in these systems generally consists of a monolayer of hepatocytes attached to either plastic or extracellular matrix protein. This standard culture configuration, suitable for most other primary cultures, is, however, inherently different from that found in the intact liver. On the one hand, as do other epithelial structures, hepatocytes possess apical and basal surfaces. However, on the other hand, unlike most epithelial cells, which have a single apical and a single basal surface, hepatocytes have a belt of apical (bile canalicular) surface that surrounds each cell and divides two basolateral (sinusoidal) surfaces, each of which is in contact with extracellular matrix. Functionally, as with all epithelial cells, the apical and basal surfaces are very different. For example, plasma proteins such as albumin are secreted across the basal surface to enter the circulation, whereas bile salts are secreted across the apical surface to enter the bile duct. It thus appears likely that, for hepatocytes to function properly, a cellular polarity reminiscent of the in vivo state needs to be maintained. In several experimental systems, it has been well demonstrated that extracellular matrix controls the formation and maintenance of cellular polarity (4,5,18,19, 23, 28, 35, 49, 50). For example, when a monolayer of

* Address correspondence to these authors. t

Harvard Medical School.

* Rutgers University.

87567938/91/3007-0237$02.50/0

mammary ductal epithelial cells cultured on collagen is overlaid with a second layer of collagen,these cells migrate to reorganize the monolayer into tubular structures (17)) thereby orienting their basal surfaces adjacent to the collagen and their apical surfaces adjacent to the lumen. In this report, we describe the effects of sandwiching hepatocytes between two layers of hydrated collagen matrix, thereby providing an environment that more closely resembles the in vivo polar geometry for the cells. Liverspecific functions and actin filament organization were maintained significantly better for hepatocytes cultured between two layers of collagen gel (sandwich system) as compared to those cultured on a single layer of collagen gel (single gel). The effects of different substrates for attachment and varying medium composition on the behavior of hepatocytes in the sandwich system are also presented. Materials and Methods Reagents and Solutions. Type IV collagenase, lot number 108F-6835,was purchased from Sigma (St.Louis, MO), Percoll from Pharmacia (Piscataway, NJ), Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose from Gibco (Grand Island, NY), fetal bovine serum (FBS), lot numbers 12103324 and 12103K30, penicillin, and streptomycin from Hazleton (Lenexa, KS) , insulin from Squibb (Princeton, NJ), glucagon from Lilly (Indianapolis, IN), epidermal growth factor (EGF) and Matrigel from Collaborative Research (Bedford, MA), hydrocortisone from Upjohn (Kalamazoo,MI), Vitrogen from Collagen Corp. (Palo Alto, CA), and Sea-Kem LE agarose from American Bioanalytical (Natick, MA). Unspecified chemicals were purchased from Sigma, Aldrich (Milwaukee, WI), EM Science (Gibbstown, NJ), JT Baker (Phillipsburg, NJ), and Mallinckrodt (Paris, KY). Perfusion buffer is 154 mM sodium chloride, 5.6 mM potassium chloride, 5.5 mM glucose, 25 mM sodium bicarbonate, and 20 mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid (Hepes),pH 7.4. Phosphate-buffered saline (PBS) is 138 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM sodium phosphate, and 1.1 mM potassium

0 1991 American Chemlcal Society and American Instltute of Chemical Englneers

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phosphate, pH 7.4. Hanks' balanced salt solution (1X HBSS) is 138 mM sodium chloride, 5.4 mM potassium chloride, 0.33 mM sodium phosphate, 0.33 mM potassium phosphate, 0.8 mM magnesium sulfate, and 5.6 mM glucose, pH 7.4. Preparation of Hepatocytes. Hepatocytes were isolated from 2-3-month-old female Lewis rata (Charles River, MA), weighing 180-220 g, by a modified procedure of Seglen (41). Animals were anesthetized in a chamber containingsaturated ether. The liver, weighing 7-8 g, was first perfused through the portal vein in situ with 400 mL of perfusion buffer with 1mM ethylenediaminetetraacetic acid (EDTA) a t 45 mL/min. The perfusate was equilibrated with 5 L/min 95% 02 and 5% C02 through 5 m of silicone tubing (inner diameter 0.058 in., outer diameter 0.077 in.) and was maintained by a 100-mm heat exchanger (reflux condenser 283000, Kontes, Vineland, NJ) a t 37 "C before entering the liver. The liver was subsequently perfused with 200 mL of 0.05% collagenase in perfusion buffer with 5 mM calcium chloride at the same flow rate. During this time, the liver swelled to about twice the original size. The swollen liver was dissected away from ligaments and the diaphragm and was transferred to a 100-mm dish with 20 mL of ice-cold perfusion buffer. The liver capsule was teased apart, and the resulting cell suspension was filtered through two nylon meshes with grid sizes 250 and 62 pm (Small Parts, Miami, FL). The cell pellet was collected by centrifugation at 50g for 5 min. Cells were further purified by a modified procedure of Kreamer et al. as follows (18). The cell pellet was resuspended to 50 mL, and 12.5 mL of cell suspension was added to 10.8 mL of Percoll and 1.2 mL of 1OX HBSS. The mixture was centrifuged at 500g for 5 min, and the cell pellet was washed twice with DMEM. Routinely, 200300 million cells were isolated with viability between 92 5% and 99 % as judged by trypan blue exclusion. Nonparenchymal cells, as judged by their size (less than 10 pm in diameter) and morphology (nonpolygonal or stellate), were less than 1% Preparation of Rat Tail Tendon Collagen. Type I collagen was prepared from Lewis rat tail tendons by a modified procedure of Elsdale and Bard (12). Four tendons were dissected from each rat tail and stirred in 200 mL of 3% (v/v) acetic acid overnight at 4 "C. The solution was filtered through four layers of cheesecloth and centrifuged a t 12000g for 2 h. The supernatant was precipitated with 40 mL of 30% (w/v) sodium chloride, and the pellet was collected by centrifugation at 4000g for 30 min. The pellet was dissolved in 50 mL of 0.6% (v/v) acetic acid, and the solution was dialyzed against 500 mL of 1 mM hydrochloric acid five times. For sterilization, 0.15 mL of chloroform was added to the solution. The solution was stirred for 2 days loosely capped to allow evaporation of chloroform. A 5-mL aliquot was lyophilized and weighed to determine the yield of collagen. Generally 100 mg was isolated per rat tail. This preparation yields type I collagen molecules mostly in its native, not cross-linked, triple-helical form (12). Hepatocyte Culture. Collagen gels were prepared by distributing 1 mL of collagen gel solution (1 part lox DMEM, pH 7.4, and 9 parts collagen solution at 1.11mg/ mL, chilled on ice, mixed just prior to use) evenly over a 60-mm tissue culture dish (Falcon, Lincoln Park, NJ) and incubated at 37 "C at least 1h before use. Collagen forms a gel at physiological pH and ionic strength at room temperature, but the rate of gelation is accelerated at higher temperature. Two million viable cells were seeded in 4

.

mL of medium, consisting of DMEM supplemented with 10% (v/v) FBS, 0.5 unit/mL insulin, 7 ng/mL glucagon, 20 ng/mL epidermal growth factor, 7.5 pg/mL hydrocortisone, 200 units/mL penicillin, and 200 pg/mL streptomycin. This constituted the single-gel system. For the sandwich system, an additional 1 mL of collagen gel solution was distributed over the cells after 1day of culture at 37 "C and 10% COZ. Culture medium was first removed and care was taken to ensure that the second layer of collagen gel was evenly spread over the entire dish. Thirty minutes of incubation at 37 "C was allowed for gelation and attachment of the second gel layer before the medium was replaced. Culture medium was changed daily. The collected media samples were stored a t 4 "C prior to analyses. For Matrigel cultures, 1 mL of thawed solution was quickly distributed over a prechilled 60-mm dish. For agarose overlay, 0.5% (w/v) agarose in PBS was boiled and brought to 37 "C before 1mL was applied to the cells for 5 min at room temperature. For floating collagen gel cultures, cells attached to collagen gel after 1day of culture were detached with a stainless steel spatula and were shaken loose from the dish (33). For experimenta with enzymatic induction, phenobarbital (Elkins-Sinn, Cherry Hill, NJ) was added to the culture medium at 2 mM. Analytical Assays. Collected media samples were analyzed for rat albumin, transferrin, and fibrinogen content by enzyme-linked immunosorbent assays (ELISA). Chromatographically purified albumin and transferrin were purchased from Cappel (Cochranville, PA), and fibrinogen was purchased from Sigma. Antibodies to the above proteins were purchased from Cappel. Antibodies specific for transferrin and fibrinogen were conjugated to horseradish peroxidase by the periodate method (48). The 96-well plates (NUNC-Immuno Plate, Maxisorp, Newbury Park, CA) were coated with 100 pL of rat albumin, transferrin, or fibrinogen a t 50,25, or 25 pg/mL, respectively, in 25 mM carbonate buffer, pH 9.6, overnight at 4 "C. The wells were washed four times with PBS plus 0.5 % (v/v) Tween 20 (PBS-Tween). Fifty microliters of sample was mixed with an equal volume of antibody (800ng/mL in PBS-Tween) before it was transferred to the wells. After overnight incubation a t 4 "C, the wells were washed four times with PBS-Tween and were developed with 100 pL of 25 mM citrate and 50 mM phosphate, pH 5, containing 0.4 mg/mL o-phenylenediamine and 0.012% (v/v) hydrogen peroxide at room temperature. The reaction was stopped with 50 pL of 8 N sulfuric acid after 7 min of incubation. The presence of bound antibodies was detected by the conversion of o-phenylenediamine by the conjugated peroxidase. The absorbance was measured at 490 nm with the Dynatech MR600 microplate reader (Chantilly, VA). Positive controls included known concentrations of purified rat albumin, transferrin, and fibrinogen added to culture medium, and negative controls included the culture medium and PBS-Tween. Concentrations of standards were calibrated by their absorbances at 280 nm, by using 0.6, 1.0, and 1.5 as extinction coefficients for 1mg/mL solutions of albumin, transferrin, and fibrinogen, respectively. Concentrations of samples were determined from a standard curve generated for each ELISA plate. Absolute rates of secretion were calculated from the concentration by multiplying the total volume of the medium (plus the volume of the collagen gel only on the day when it was introduced to the culture) and dividing by the elapsed time. Results were given in micrograms per hour per dish. Generally, two duplicate wells were averaged for each sample, and replicate cultures

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agreed within 10% of each other. Experiments repeated at least three times gave similar qualitative trends, but the absolute value varied up to 2-fold. Total bile acids were measured by the conversion of 3a-hydroxy bile acids into %oxo bile acids catalyzed by 3a-hydroxysteroid dehydrogenase, based on the method of Maahige et al. (29). Ureanitrogen content was measured by the specific reaction of diacetyl monoxime with urea, based on the method of Crocker (7). Concentrations of samples were determined from standard curves generated with known concentrations of bile salts or urea. Background control was the culture medium alone. Reagents and standards for the bile acid and urea assays were purchased as diagnostic kits from Sigma. Experiments repeated at least three times reproduced similar results. For enzymatic detoxification, 24 pL of 250 mM p-nitroanisole in dimethyl sulfoxide was added to tubes containing cells scraped from one culture dish and 3 mL of complete medium buffered with 20 mM Hepes to measure maximal activity of P450 enzymes (45).Tubes were shaken for 1 h at 37 OC. Reaction was stopped by adding 3 mL of ice-cold 0.9% sodium chloride and 200 mM Tris, pH 9. Tubes were subsequently centrifuged at lO00g for 5 min. Two milliliters of supernatant was transferred to a cuvette to determine its absorbance at 400 nm, giving the concentration of free p-nitrophenol. Five hundred units of j3-glucuronidase (type H1, Sigma) in 0.5 M Tris, pH 5,and 175 pL of 1 N hydrochloric acid were added to the cuvette for 90 min of incubation at room temperature. One hundred and fifty microliters of 1 N sodium hydroxide was added before absorbance at 400 nm was determined, giving the concentration of total p-nitrophenol, Background controls, including cells incubated with dimethyl sulfoxide and medium incubated withp-nitroanisole,were concurrentlyperformed with each experiment. Concentration was determined from a standard curve generated from known concentrations of p-nitrophenol added to the incubation medium. Actin Localization. Cultured hepatocytes were rapidly fixed in 200 mL of 2 % (w/v) paraformaldehyde, 60 mM L-lysine, 10 mM sodium periodate, and 37.5 mM sodium phosphate, pH 7, at room temperature for 1 h (31). After overnight fixation at 4 OC, cultures were detached from the dish by gently scraping the collagen gel with a stainless steel rod. Detached gels were permeabilized with 0.1 % (v/v) Triton in PBS at room temperature for 10 min and subsequently washed four times in PBS with agitation. An equal volume of FITC-phalloidin (3.3 pM in methanol, Molecular Probes, Eugene, OR) diluted 1 5 0 with PBS was applied to the detached gel for 1h at 37 "C in a humidified incubator. Cultures were then washed four times with PBS and were mounted on a glass slide with a drop of 90% (v/v) glycerol and 0.1 mg/mL p-phenylenediamine in PBS, pH 8. For intact liver analysis, 60 mL of the same fixative was perfused through the portal vein at room temperature. After fixation, tissue was transferred to 0.6 M sucrose in PBS for overnight. The tissue was immersed in TissueTek OCT embedding medium (Miles, Elkhart, IN) for 5 min and was subsequently frozen in liquid nitrogen. Tenmicrometer cryosections were prepared on a ReichertJung 2800 Frigocut (Cambridge Instruments, Deerfield, IL) at -20 "C and were transferred onto glass slides precoated with 1mg/mL poly(L-lysine). Sectionswere treated with acetone and were permeabilized with 0.1 % Triton in PBS. The slides were then processed the same way as described above for cultures. Slides were examined on a MRC500 scanning laser confocal microscope (BRL, Rich-

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Figure 1. Rates of protein secretion, determined from collected culture media by ELISA. Albumin (A), transferrin (B),and fibrinogen (C) secretion rates were significantly higher for the sandwich system as compared to the single-gel culture. Error bars represent standarddeviationof four repeatedmeasurements from a batch of cells.

mond, CA), and pictures were taken from a black-andwhite, high-resolution monitor.

Rssults Hepatocyte Characterization in Single-Gel a n d Sandwich Culture Configurations. Protein Secretion. The culture conditions chosen were similar to those commonly employed in the literature. A range of liverspecific functions was examined to determine the functional differences between hepatocytes that were sandwiched between two layers of collagen gel and those cultured on a single layer of collagen gel. The secretory rates of albumin, transferrin, and fibrinogenwere measured over the course of 6 weeks of culture (Figure 1). Secretion rates for all proteins were found to be significantly higher for the sandwich system as compared to the single gel. For the single gel, albumin and fibrinogen secretion rates followed a course of decline during the first week of culture; the transferrin secretion rate increased slightly before the onset of decline starting on day 5. For the sandwich system, there appeared to be a 2-3-week period before maximal, steady-state protein secretion rates were achieved. The content of total DNA of the two culture systems were not significantlydifferent by the end of 1week (11);totalDNA thereafter was stable for the sandwich system, whereas total DNA continued to decrease for the single gel. Bile Salt and Urea Production. To assess the competency of the secretion machinery across the apical

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Figure 2. Rates of bile salt and urea secretion,determined from collected culture medium by enzymatic assays. Bile salt (A) and urea (B)secretion rates were significantly higher for the sandwich system as compared to the single-gel culture. Error bars represent standard deviation of four repeated measurements from a batch of cells.

surface, total bile salts were measured in the collected media. The secretion rates were found to be similar for the two systems during the first week (Figure 2A). Thereafter, however, the secretion rate for the single gel decreased rapidly, whereas the secretion rate remained relatively constant for the sandwich system. Hepatocytes are intimately involved with metabolism of carbohydrates,lipids, and amino acids. These generally involve cytoplasmic enzymes that are liver-specific,such as the conversion of ammonia to urea by the urea cycle enzymes. The rate of urea production was found to be high for both systems initially (Figure2B). The production rate stabilized after 2 weeks for the sandwich system but continued to decrease for the single gel. In summary, the sandwich system performed significantly better than the single gel for all of the measured liver-specific functions. Enzymatic Detoxification. It had been shown previously that hepatocytes cultured on a single layer of collagen gel are capable of enzymatic detoxification within 1week of culture (34). To demonstrate that hepatocytes cultured in the sandwich system are also capable of enzymatic detoxification, the induction of P450 enzymes by phenobarbital (PB) was measured by using 2 mM p-nitroanisole (45).Comparisons were made between cultures that were treated without PB, with PB, and with PB after a period of culture without PB. In the absence of PB, enzymaticactivity of hepatocytes decreased rapidly in the first week (Figure 3). In the presence of PB, the enzymatic activity was significantlyhigher during the first week. After the enzymatic activity had become low in cultures without PB, it was possible to dramatically increase the activity by culturing these cells with PB. Therefore, the activity of the P450 enzymes correlated well with the addition of the inducer; in the absence of PB,the enzymatic activity rapidly dropped, and in the presence of PB, the enzymatic activity quickly returned. Actin Distribution. To demonstrate the effect of different culture configurations on cytoskeletal organization, the distribution of intracellular actin was localized by phalloidin (13). In the liver, hepatocytes possessed

Figure 3. Rates of p-nitroanisole conversion for the sandwich system, determined spectrophotometrically. Free p-nitropheno1 was first measured, and the conjugated portion was enzymatically cleaved to yield total conversion of p-nitroanisole. In the absence of phenobarbital, the enzymatic rate decreased rapidly. A higher enzymatic rate was maintained in the presence of phenobarbital. When phenobarbital was added for 4 days after 4 or 8 days of culture without phenobarbital, the enzymatic rate returned to a high level. Error bars represent standard deviation of measurements from three batches of cells.

** Figure 4. Micrograph taken on a confocal microscope showing the distribution of actin in normal liver revealed by the binding of FITC-phalloidin. Actin filaments were localized beneath the plasma membrane and were particularly concentrated around the bile canaliculi between two cells (arrows). The nucleus of a hepatocyte is denoted N, and the sinusoid is denoted S. Repeated experiments with different livers reproduced similar results.

sub-plasma-membrane actin that is particularly dense around the bile canaliculus (Figure 4). After 1 day of culture on a single layer of collagen gel, hepatocytes possessed a distribution of actin similar to the in vivo state (Figure 5A), except that the cellular surface adjacent the collagen gel showed the beginning stages of stress fiber formation (Figure 5B). The size of the cells appeared to be larger than that in the normal liver, suggesting some spreading of the cultured cells. A t 7 days on a single layer of collagen gel, hepatocytes increased the quantity of stress fibers so extensively that the entire cytoplasm appeared to be filled with actin filaments (Figure 5C). Hepatocytes cultured for 7 days in the sandwich system, on the other hand, possessed an actin filament distribution similar to that seen in normal hepatocytes and showed no signs of stress fiber formation (Figure 5D). Effect of Matrix and Medium on the Sandwich System. Reversibility ofthe Single Gel Culture. After 1 week of culture, hepatocytes cultured on a single layer of collagen gel secreted albumin, transferrin, and fibrinogen relatively poorly (Figure 1). Upon the addition of asecond layer of collagen gel a t that time, such cells dramatically increased the secretory rates of albumin, transferrin, and

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Figure 5. Micrographs taken on a confocal microscope showing the distribution of actin filaments revealed by the binding of FITCphalloidin. (A) Hepatocytes were cultured for 1 day on a single layer of collagen gel. An optical section was taken approximately 8 pm away from the cell attachment to the collagen gel. The distribution of actin was similar to that of the normal liver. Arrows point at the presumed bile canaliculi. (B) This optical section was taken from the same position as that in (A) but approximately 4 pm away from the cell attachment to the collagen gel. Close to the attachment, the formation of stress fibers (arrows) can be observed in these cultured cells. (C) Hepatocytes cultured for 7 days on a single layer of collagen gel. This optical section was taken approximately 6 pm away from the cell attachment to the collagen gel. This and all other optical sections showed extensive formation of the stress fibers (arrows). (D)Hepatocytes cultured for 7 days in the sandwich system. This optical section was taken approximately 6 pm away from the cell attachment to the bottom collagen gel. This and all other optical sections showed an actin distribution similar to that of the normal liver. Arrows point at the presumed bile canaliculi. Repeated experiments with different batches of cells reproduced similar results.

fibrinogen to levels similar to those of hepatocytescultured in the sandwich system initially (Figure 6). For urea and bile acid secretion, rates for cells cultured in the single-gel system were comparable to those in the sandwich system by the seventh day (Figure 2). When a second layer of collagen was added to cells in the single-gel system on the seventh day, the rates of urea and bile acid secretion remained similar to those in the sandwich system (data not shown). Therefore, the second layer of collagen gel could be added up to 7 days after hepatocytes had been cultured in the single gel to reverse the deteriorating course of cell function. Effect of Different Matrix Substrates. The effect of the collagen gel sandwich could be the result of physicochemical changes (e.g., protection from shear stress, retention or exclusion of molecules) or biological changes (activation of receptors, organization of complex extracellular matrix and cytoskeleton). In order to better define the properties of the rat tail tendon collagen hydrogel necessary for supporting hepatocyte culture in the sandwich configuration, different collagenous and noncollagenous materials were employed. These substrates include Vitrogen, agarose, and plastic. Secretion of albumin was used as a marker for functional performanceof the cultured cells. Vitrogen, a commercial preparation of bovine skin type I collagen, was found to work as well as the rat tail tendon collagen in the sandwich configuration (data not shown). Thus, there appeared to be no specific requirement for collagen from the same species. To test whether

attachment to any substrate would be necessary and sufficient, agarose and plastic surfaces were used. When hepatocytes were seeded on agarose, they did not attach and remained in suspension, whereas hepatocytesattached rapidly to tissue-culture plastic. When agarose was overlaid on hepatocytes that were cultured on a single layer of collagen gel, a transient maintenance of albumin secretion was observed (Figure 7). When hepatocytes cultured on plastic were overlaid with collagen gel, these cells secreted similar amounts of albumin as those in the sandwich collagen gels (data not shown). Therefore, the exact composition of the material for constructing the sandwich system appeared to be unimportant as long as cellular attachment to that substrate was possible. Two systemsdescribed in the literature were reproduced in our laboratory for the purpose of comparison: Matrigel, an extract of the complex matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (2,3,37),and floating collagen gel (33). Albumin secretion was used as a marker for comparison. The results from cells cultured on Matrigel were similar to those obtained when hepatocytes were cultured in the sandwich system (Figure 7). As compared to the floating collagen gel, where a single layer of collagen gel cultured with hepatocyteswas allowed to float, hepatocytes cultured in the sandwich system functioned significantly better. Effect of Media Composition. Since the formulation of the culture medium was developed by other investigators who employed plastic dishes to culture hepatocytes (3),it

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Figure 7. Different substrata composition and configuration modulated the secretionof albumin as measured by ELISA. Hepatocytes overlaid with agarose maintaineda transient period of albuminsecretion,which was better than cells cultured on a single layer of collagen gel. Hepatocytesculturedon Matrigel performed equally well as those cultured in the sandwich system. Hepatocytes cultured on the floating collagen membrane maintained a low rate of albumin secretion over a sustained period. Result represents the average of two replicate dishes from one batch of cells. Repeated experiments reproduced similar qualitative results.

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14 21 DAYS Figure 6. Rates of protein secretions for the sandwich system (double gel, day 1) and the converted system (double gel, day 7), determined from collected media by ELISA. After a period of decrease,the secretion rates of albumin (A),transferrin (B),and fibrinogen (C) in the converted system increased rapidly to levels similar to those of the sandwich system. Error bars represent standard deviation of four repeatedmeasurementsfrom one batch of cells.

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is possible that some of the medium supplements are no longer necessary for the sandwich system. In the next set of experiments, medium composition changes were made in order to define critical components for hepatocyte culture in the sandwich system. For hepatocytes cultured in the sandwich system, removal of hydrocortisone or serum resulted in significant changes in the secretion of albumin (Figure8A). In the absence of serum, the maximal rate of albumin secretion appeared to be greater than that achieved in its presence. In serum-free cultures, removal of insulin and epidermal growth factor decreased the maximal rate of albumin secretion, whereas the removal of glucagon increased the maximal rate. Removal of hydrocortisone,regardleasof the presence or absence of serum supplement, resulted inverylow levels of albumin secretion and stellate morphology of the hepatocytes. This effect can be prevented by adding hydrocortisone at 75 ng/mL (Figure 8B). It is customary to supplement hepatocyte culture medium with a high concentration of insulin (3, 40). Typically,the insulin concentration of the culture medium is 3-4 orders of magnitude higher than that of normal serum concentration (6-26 microunits/mL). However, high levels of insulin in the culture medium led to fatty changes readily observed as lipid droplets in the cytoplasm of hepatocytes cultured in the sandwich system. By ti-

trating the amount of insulin addedto the culture medium, it was found that fatty changes no longer occurred when insulin was near normal serum concentration (data not shown),and there was essentially no effect on the secretion of albumin (Figure 8C). The omission of other hormone additives, except for hydrocortisone mentioned above, did not result in major effects on morphology or total DNA content. In summary, the exact composition of the culture medium appeared to be unimportant in maintaining cell viability in the sandwich system if normal serum levels of hormones were present; however, the composition of the medium did influence the quantitative rate of albumin secretion.

Discussion These results taken together suggest that hepatocytes sandwiched between two layers of collagen gel are capable of performing a wide variety of cellular functions normally present in the liver. Hepatocytes cultured on asingle layer of collagen gel for 7 days can be converted into a sandwich system, as shown by reversibility of cellular function upon addition of a second layer of collagen gel. The sandwich system as compared to the best available system, Matrigel, performed equally well. Furthermore, other materials to which hepatocytes can attach will likely work the same way as does collagen gel. Within the sandwich system, there appears to be no critical requirement for specific factorsother than physiologicalconcentrationof hormones. In the two experimental systems, single gel and sandwich, the only difference is the additional layer of collagen gel. This relatively simple addition produced dramatic effects both morphologically and functionally. Conceptually, the collagen gel overlay may exert one or more of the following effects: it may act as a physical barrier to reduce mechanical stress on cell membranes, it may alter cytoskeletal organization such that the correct cellular polarity or shape can be established, it may allow the organization of a complex extracellular matrix network that secondarilyaffectsgene expression,or it may organize cell-cell contacts such that direct cell-cell communication is optimal. The results presented above do not formally distinguish among these possibilities. Some elements of all four processes are evident. For example, when agarose, a substrate to which hepatocytes do not attach, was

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Figure9. Schematicrepresentation of actin f i i e n t distribution in the cultured hepatocytes. For the single gel, actin filaments were constrained to form bundles of stress fibers at the base of the cell near the attachment. For the sandwich system, actin filaments attached from one side of the cell surface traversed around the bile canaliculus to connect to the other side of the cell surface.

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DAYS Figure 8. Alterations in the additives to the culture medium influenced the rate of albumin secretion in the sandwich system as measured by ELISA. (A) Hepatocytes maintained high rates of albumin secretion in serum-free medium. The additional omission of hydrocortisone significant affected the secretion of albumin. (B)The necessary hydrocortisoneconcentration in the completemedium to maintain albumin secretionis closeto normal serum concentration(&250ng/mL). (C)The omission of insulin from the complete medium had minimal effect on the secretion of albumin. Result represents the average of two replicate dishes fromone batch of cells. Repeated experimentsreproducedsimilar qualitative results.

overlaid on hepatocytes cultured on a single layer of collagen gel, a transient maintenance of albumin secretion was observed. The overlyingagarose may serve as a barrier to mechanical stress. This effect, however, is much more pronounced when the support is a surface to which hepatocytes can attach, although the exact composition of the support appears to be unimportant, as shown by the experiment where plastic was substituted in place of collagen gel on one side of the hepatocytes. Conceivably an adhesive, thin, macroporous, rigid support could be developed in lieu of the collagen gels. However, collagen gels do have many advantages: pure collagen is readily

available in large quantity it is relatively inexpensive, it is biologically compatible, and it can be easily overlaid on the attached cells. An alternative way to construct the sandwich system is to mix a hepatocyte suspension with collagen solution that is in the process of gelation (24)and allow the hepatocytes to settle in a monolayer before gelation is completed. This technique sandwiches a sheet of hepatocytes between collagen gel in one step. The effect of the collagen gel overlay also correlates with a change in the cytoskeletal organization of the cultured hepatocytes. This was demonstrated with actin localization by phalloidin. For hepatocytes cultured on a single layer of collagen gel, the formation of stress fibers could be seen on the first day and progressively increased with culture time. For hepatocytes cultured in the sandwich system, no formation of stress fibers was observed, which corresponded to the in vivo state. Since hepatocytes in the sandwich system were cultured on a single layer of collagen gel on the first day, rearrangement of actin filaments within the cell must have occurred upon the addition of the collagen gel overlay. This suggests a casual relationship between the extracellular matrix configuration and the organization of the cytoskeleton. This is not surprising since actin is linked through a series of membrane proteins includingtalin, vinculin, and fibronectin receptor to the extracellular matrix. One could postulate that the formation of the stress fibers on a single layer of collagen gel is a direct result of the culture configuration. Since actin filaments attach from focal contact to focal contact, hepatocytes attached to a one-sided support invariably end up with stress fibers on one side of the cell (Figure 9). For cells cultured within a sandwich configuration,focal contact to focal contact can be oriented across the hepatocytes so no stress fiber forms. The space of Disse, the extracellular space between hepatocytes and the endothelial cells, contains multiple extracellular matrix proteins including type I collagen (30). Furthermore, hepatocytes can synthesize extracellular matrix components in vitro (6,10,44), which we have also confirmed for the sandwich hepatocytes (data not shown). It is possible that collagen gels act as a scaffold that allows the incorporation of newly synthesizedextracellularmatrix materials in the proper configuration. While some investigators report complex extracellular matrices to be a part of the necessary starting ingredient for long-term our experience cultures of hepatocytes (I, 2,39,42,43), with the collagen gel sandwich system suggests otherwise. Direct comparison to results reported in the literature with other culture systems is difficult because of differences in strain and sex of animals used, conditions of cell isolation, methods for cell purification, conditions of cell

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culture, and methods for assays. The results of comparison studies performed in our laboratory involving two other methods for long-term hepatocyte culture showed that the sandwich system performed as well as the Matrigel system and significantly better than the floating collagen gel system. Although hepatocytes cultured on Matrigel appeared to be stable phenotypically, this culture system suffers from many disadvantages. Hepatocytes cultured on Matrigel cluster into multicellular spheroids, sometimes connected to one another forming a three-dimensional and individual cells do not exhibit the normal lattice (1,2), polygonal shapes typical of hepatocytes in vivo. Furthermore, Matrigel is a complex mixture of extracellular matrix proteins with many undefined components, and the preparation of Matrigel results in significant batchto-batch variations. Hepatocytes cultured in the sandwich system, on the other hand, exhibited the familiar polygonal morphology, and the preparation of type I collagen is quite pure and easily available in large quantity. The measured rates of protein secretion of hepatocytes in the sandwich system can be compared to those reported for the normal intact liver. Such a comparison, however, should not be given too much significance since the cultured hepatocytes experience an artificial environment somewhat different from the plasmalike fluid that bathes hepatocytes in vivo. The rates of protein synthesis had been estimated to be 0.74,0.14, and 0.06 mg h-’ (g of liver)-l for albumin, transferrin, and fibrinogen, respectively (22, 36,38). If one assumes 100 X 106 hepatocytes in 1 g of liver, the synthesis rates are equivalent to 7.4,1.4, and 0.6 pg h-1 104 cells for albumin, transferrin, and fibrinogen. These values are fairly close to the observed rates of protein secretion. The measured fibrinogen secretion may be an underestimate of the actual rate, since some of the fibrinogen secreted may have clotted and become attached to the collagen gel (44). The reversibility of the degenerative changes that occurred in hepatocytes cultured on a single layer of collagen gel was demonstrated by the ”rescue” experiments. Hepatocytes cultured on a single layer of collagen gel gradually lost function and eventually died and detached from the dish. However, up to a period of 1week, the fate of these cells can be reversed by the addition of collagen gel overlay, thereby converting the culture into a sandwich system. Immediate improvement of function can be observed after 1day of collagen gel overlay. This suggests that hepatocytes cultured on a single layer of collagen, for up to 7 days, undergo changes that are rapidly reversible, and the timing of the change correlates with the addition of the collagen overlay. Although in Figure 6 the rescued cells appeared to “overshoot” the protein secretion rates compared to the sandwich system, this was not always the case. There appeared to be a considerable variation with respect to this aspect of behavior. With roughly equal frequency, rescued cells can also “undershoot” the secretion rate of the sandwich system. Due to the elevated hormone concentration in the portal vein and the catabolic activity of the cells, hormonal additives are often employed a t high concentrations for the culture of hepatocytes (3,40). The formulation of the culture medium is generally not designed to optimize longterm cell function. In this study, it was shown that hepatocytes in the sandwich system can be cultured in a serum-free environment, and no single additive, with the exception of hydrocortisone, exerts any significant effect with respect to long-term maintenance of hepatocyte function in serum-free media. The concentration of hy-

Bbtechnol. Rog..,1991, Vol. 7, No. 3

drocortisone, however, only needs to be in the physiological range (50-250 ng/mL). The data presented demonstrate that hepatocytes sandwiched between two layers of collagen gel maintain multiple liver-specificfunctions for at least 42 days. This culture system does not require complex extracellular matrix molecules or specific factors in the medium; however, the additional layer of collagen gel may pose practical constraints for the purpose of experimentation such as pulse-chase labeling. The mechanism for functional maintenance in the sandwich system is unclear, but it is likely to be related to the induced polarization of the hepatocytes. A reversible change in the organization of the cytoskeleton is found to correlate with a change in the configuration of the extracellular matrix. This simple culture system can serve as an in vitro model for the studies of long-term hepatocyte function or as a basis for the development of an artificial liver.

Acknowledgment We thank Robert M. Ezzell for his assistance with confocal microscopy experiments, Michael D. Harmon, Lauren H. Kim, Eve J. Smith, and Laura M. Sterling for their technical assistance, and Karen Ukleja for her assistance in preparation of the manuscript. This work was supported by NIH Grants DK-01746 and DK-41709 and a grant from Shriners Hospitals for Crippled Children (SHCC). M.L.Y. is a Lucille P. Markey Scholar in Biomedical Science. J.C.Y.D. is a National Science Foundation fellow.

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